The language "inactivation of virus", or "substantially free of infectious virus" as used herein means that the residual titer of biologically infectious virus in products treated according to the method described below is so low that infusion of therapeutic quantities of the product into a plurality of normal animal hosts for the virus in question will not produce statistically significant clinical or serological evidence of infection (p&lt;0.1) in a statistically significant host population. This does not require that the products be sterilized, or completely free of virus, because compositions having a low viral titer may have therapeutic utility where the alternative is a more contaminated material.
Hageman factor, prothrombin and factors XI, VII, IX and X are all plasma proenzymes. They participate in a complex cascade of enzyme mediated reactions culminating in the conversion of fibrinogen to a fibrin clot. Their participation in this cascade is initiated by changes in the primary or conformational structure of the proenzyme which converts it to an active proteolytic enzyme. This change is termed "activation" of the proenzyme. For the sake of brevity, "blood clotting enzymes" shall include both the enzymatically nonfunctional proenzymes as well as their enzymatically active forms obtained by activation. Similarly, recitation of factor designations or names as used herein shall be construed to include both forms of the enzymes, e.g., "factor X" shall include both activated as well as unactivated factor X and mixtures thereof. From time to time the abbreviation "F" will be used for "factor".
Each of the blood clotting enzymes has its own specificity and performs an established task in the clotting mechanism. For example, activated factor IX hydrolyzes factor X in the presence of calcium and factor VIII, a protein cofactor, to produce activated factor X. Thrombin (activated prothrombin) cleaves fibrinogen, a protein substrate, to yield fibrin monomers which eventually form a clot.
Factors VII (proconvertin), IX (plasma thromboplastin antecedent) and IX (Christmas factor) are all beta globulins. The molecular weight of factors VII and IX are about 35,000 and 50,000 respectively. Factor X, also called Stuart-Prower factor, is an 87,000 molecular weight alpha globulin. Prothrombin (factor II) is a glycoprotein having a molecular weight of about 68,000. Hageman factor (factor XII) is also a glycoprotein, but its molecular weight is about 82,000.
These factors all have in common their biological role as proenzymes and clotting enzymes in the clotting of blood, a role not shared by other plasma proteins such as antithrombin III, albumin, antihemophilic factor (factor VIII), gamma globulin or fibrinogen. Plasminogen is not a blood clotting proenzyme as that term is used herein because its active form does not participate in the generation of the clot but instead digests fibrin in an existing clot.
Blood clotting enzymes are prepared commercially by known methods from large pools of individual blood plasma donations. All plasma units utilized in their manufacture are tested for the presence of hepatitis B surface antigen using test systems licensed for that purpose. These immunological tests, however, are not sufficiently sensitive to detect all potentially infectious units of hepatitis B. In addition, hepatitis virus may be concentrated during the fractionation procedure. Thus, the small amount of undetectable virus that may be contained in a large plasma pool may become significantly infective when it is concentrated several fold.
The development of specific diagnostic test for hepatitis A and hepatitis B has made it possible to identify a third type of viral hepatitis that is apparently unrelated immunologically to either of the first two. The transmissible agent, non-A, non-B hepatitis, has been confirmed by transmission of the disease to chimpanzees by exposure to pathological material. There are several lines of evidence pointing to more than one non-A, non-B hepatitis virus. These include cases with sequential episodes of apparent acute non-A, non-B hepatitis, variability in epidemiology and clinical syndromes including incubation periods.
Specifically in the case of blood clotting enzymes, hepatitis transmission via infusion of the products remains a problem even where the source plasma has been screened for hepatitis B. (Roberts et al., "Thrombos. Diathes. Haemorrh. [Stuttg.] 33: 610-616 [1975]). Furthermore, it is conceivable that adventitious viruses other than those responsible for hepatitis could contaminate the preparations. Thus a need exists for a process that will inactivate all viruses, including hepatitis, that may be found in these preparations.
Scattered reports exist of inactivating hepatitis virus in plasma or solutions of blood plasma fractions by heating the liquid plasma or fraction. Murray in "The New York Academy of Medicine" 31(5): 341-358 (1955) reports inactivating icterogenic activity in plasma by heating at 60.degree. C. for ten hours. This procedure has long been used to inactivate hepatitis in albumin and plasma protein fraction (PPF) solutions.
Belgian Pat. No. 844,566 discloses heating liquid plasma or serum for 1/2-4 hours at 50.degree.-60.degree. C. to kill hepatitis virus, followed by fractionating the plasma or serum to obtain immune globulin G.
West German Offenlegungschrift No. 29 16 711 discloses stabilizing plasminogen, prothrombin, antithrombin III and factors II and VIII against heat in aqueous solution by adding an amino acid and a monosaccharide oligosaccharide or sugar alcohol to the solution. The solutions were heated for 1 minute to 48 hours at from 30 to 100.degree. C. to inactivate hepatitis virus.
All of the foregoing techniques are disadvantageous. While the viruses may be inactivated, certain therapeutic proteins are also inactivated because they are thermally unstable in aqueous solution. Thus a reduction in the infectiousness of such protein compositions is accompanied by losses in the biological activity of the protein.
It is known to inactivate canine hepatitis virus in dry fibrinogen or albumin preparations by heating the preparations for 10 hours at 60.degree. C. (Rozenberg et al. XII International Congress on Blood Transfusion, Abstracts p. 473-474 [1969]).